The use and abuse of a class of illicit designer drugs known commonly as “ecstasy-class drugs” have increased significantly in recent years. These compounds, which are derivatives of amphetamine distinguished by having a fused methylenedioxy phenyl ring system, include MDA (3,4-methylenedioxyamphetamine), MDMA, also known as “Ecstasy” (3,4-methylenedioxy-N-methylamphetamine), MDEA, also known as “Eve” (3,4-methylenedioxy-N-ethylamphetamine), BDB (3,4-methylenedioxyphenyl-2-butanamine), and MBDB (3,4-methylenedioxyphenyl-N-methylbutanamine).
Heretofore, methods for the detection of ecstasy-class drugs have primarily involved immunoassays originally developed for the detection of amphetamine and/or methamphetamine. The detection of an ecstasy-class drug by such assays relies on the limited cross-reactivities that may coincidentally exist between the ecstasy-class drug and the amphetamine and/or methamphetamine antibodies. A positive result obtained by such an assay may still not indicate which particular substance or member of the methylenedioxy (MD) class of derivatives is present in a sample.
In general, amphetamine and methamphetamine immunoassays are relatively insensitive to, and non-specific for, ecstasy-class drugs. Such assays show particularly limited recognition for the MDEA derivative.
The present invention is directed to remedying these and other problems relating to the use of conventional amphetamine and/or methamphetamine immunoassays for the detection of members of the methylenedioxy class, or ecstasy-class, drugs.
In testing for drugs of abuse, immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as β-galactosidase and peroxidase, fluorescent molecules such as fluorescein compounds, radioactive compounds such as 125I, and microparticles are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
Currently popular are automated assays based on kinetic interaction of microparticles in a solution as measured by changes in light transmission. In the absence of sample drug, free antibody binds to drug-microparticle conjugates causing the formation of particle aggregates. As the aggregation reaction proceeds in the absence of sample drug, absorbance increases. When a sample contains a drug in question, the drug competes with a particle-bound drug derivative for free antibody. Antibody bound to sample drug is no longer available to promote particle aggregation, and subsequent particle lattice formation is inhibited. The presence of sample drug diminishes increasing absorbance in proportion to concentration of drug in the sample. Sample drug content is determined relative to the value obtained for a known cutoff concentration of drug.
A problem in assays for amphetamine-class drugs is that prior art methods do not provide sufficient cross-reactivity with and displacement from antibodies to measure all the amphetamine-class analytes in a sample, especially amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methylamphetamine (MDMA), and 3,4-methylenedioxy-N-ethylamphetamine (MDEA), with sufficient sensitivity. The methamphetamine derivatives of the present invention overcome such problems by providing the required cross-reactivity with and displacement from methamphetamine antibodies to sensitive measurement of amphetamine, methamphetamine, and ecstasy-class compounds.
Another problem overcome by the present invention is that of unwanted conjugation of haptens through the free amino group on the methamphetamine hapten. The derivatives of the present invention avoid this problem.
In U.S. Pat. No. 5,135,863 issued Aug. 4, 1992, Hu et al. describe conjugates comprising a label bound to an analog of amphetamine or methamphetamine via a thiol linkage. The conjugates further comprise a linker bonded to the benzene ring at the meta- or para-position, and the amphetamine nitrogen is unprotected.
In U.S. patent application Ser. No. 2001/0051158 published Dec. 13, 2001, Owens et al. describe immunochemical haptens which are meta-position derivatives of methamphetamine. The free amino group on the methamphetamine hapten is unprotected.